Naphthalimide-Based Template for Inhibitor Screening via Cross-Linking and In-Gel Fluorescence: A Case Study against HCA II

Abstract

We describe a rapid electrophoresis-based method for profiling of carbonic anhydrase inhibitors. In addition to the pharmacophore moiety intended for reversible interaction with a target enzyme, a fluorescent template with a built-in azide group for photoaffinity labeling is also included as a part of the inhibitor design. Following incubation and irradiation, gel electrophoresis with visualization under UV allows assessment of the efficiency of cross-linking. The relative efficiency of cross-linking of various probes can be regarded as a reflection of their inhibition potencies, an assumption supported by the trend in their IC₅₀/K_i values. The method has the advantage of being applicable to impure enzyme preparations and also can be used to screen several inhibitors including their promiscuity in parallel in a short time as has been currently demonstrated with HCA II.

Introduction

Identification of target receptors/enzymes whose over- or underexpression is often correlated with a disease plays a primary role in the drug discovery process.¹ Once the target is identified, the classical screening can be performed for small-molecule libraries against a purified form of these receptors or enzymes using in vitro assays.² However, this approach usually provides little information about target selectivity and interaction with potential off-targets in a complex proteomic mixture present in the system (Figure 1). It would be more convenient if the assay can be carried out in whole cell lysates, which not only provides information about the affinity of the small molecule substrates toward the protein but also reveals any off-target interactions. In this respect, affinity-guided targeting of a protein of interest in a mixture has emerged as a viable technique.³ In the 1960s,⁴ affinity-guided probes have a long history since their inception. The technology was recently applied for the identification of irreversible inhibitors by Bogyo and co-workers⁵ using radio-labeled probes and a subsequent electrophoresis-based assay. This rapid screening approach resulted in the identification of a cathepsin B-selective inhibitor.⁶

Figure 1.

Classical method of potential inhibitor screening of a mixture of proteins A, B, C, and D.

More recently, Li et al.⁷ used activity-based protein profiling (ABPP) for competitive profiling to screen libraries of carbamates as irreversible inhibitors of uncharacterized serine hydrolases. Subsequently, Cravatt et al.⁸ have successfully applied the completive affinity-based technique for screening of reversible inhibitors using a competitive fluorescent rhodamine-based assay (Figure 2). The advantage of this method is that promiscuous inhibitors lacking specificity can be readily rejected without any further processing. The method, though ingenious and novel, can be technically challenging as it requires previous knowledge of the kinetics of binding.

Figure 2.

Cravatt’s strategy for screening of reversible inhibitors.

A comparatively straightforward method would be to have a fluorescent photo-cross-linker part (acting as a reporter) included in the inhibitor design by attaching it to a possible pharmacophore. In that case, the reversible enzyme–inhibitor complex can be permanently bonded to the target enzyme via photoirradiation. Subsequent gel electrophoresis of the photo-cross-linking experiment followed by visualization, for example, under UV and using Coomassie blue, should indicate the efficacy of the inhibitor. However, one issue could be the combined size of the fluorophore and the photo-cross-linker, which may weaken the binding. For this not to happen, one needs to use a photoaffinity group-embedded fluorescent label of smaller steric size as compared to the frequently used ones like rhodamine/fluorescein (Figure 3). To have a minimal effect of the reporter moiety on the pharmacophore activity, this has been kept apart by a linker of optimized length (reported earlier⁹) with three methylene groups. Keeping this in mind, we have designed and synthesized a series of naphthalimide–aryl sulfonamide hybrids (1–11) and have shown that it is possible to compare their relative inhibition potencies by their relative cross-linking efficiencies. The method is validated by doing the cross-linking experiment against a control compound (vide infra) as well as by comparison of IC₅₀/K_i values. The design, synthesis, and screening of potential inhibitors are described in this paper in detail.

Figure 3.

Strategy for inhibitor screening described in this paper.

Results and Discussion

Our starting point was the recently described⁹ linker-based azidonaphthalimide template, which serves three functions simultaneously. It has the in-built photo-cross-linker in the form of an azide, a less sterically bulky naphthalimide moiety as a fluorescence template, and a variable linker ending up in the carboxylic acid functionality. This 3-in-1 template has been shown to considerably improve the utility of template-based affinity probes. It could be easily connected to the selectivity hands that are potential reversible binders of human carbonic anhydrase II (HCA II)¹⁰ or penicillin-binding proteins (PBPs).¹¹ We were able to detect the presence of these enzymes in the presence of other proteins as well as in cell lysates.

To expand the scope of this 3-in-1 template, we attached various zinc binding motifs such as sulfonamide and its derivatives, carboxamide, terpyridine, and hydroxamate to produce the inhibitor molecules (structures 1–11 as shown in Figure 4). Our design was also inspired by the report by Supuran et al.¹² of excellent inhibition shown by a naphthalimide–sulfonamide hybrid (12) (K_i in a low nanomolar level). As a positive control for our photo-cross-linking studies, we synthesized the azido version (13) of Supuran’s molecule.

Figure 4.

Desired inhibitors.

Synthesis of the target inhibitors started with the GABA-attached azido naphthalimide as depicted in Scheme 1. Sulfonamides 1–5 and 7–9 and carboxamide 6 were synthesized by esterification with the corresponding bromoacylated derivatives. Hydroxamate 11 was prepared via nucleophilic displacement of the ethyl ester with free hydroxylamine.¹³ For the synthesis of terpyridine derivative 10, the 4-methyl phenyl terpyridine was first brominated,¹⁴ which was then esterified with the GABA-naphthalimide carboxylic acid. The control compound 13 was prepared via direct imide formation from azidonaphthalic anhydride and aminomethyl benzene 4-sulfonamide. All the compounds were isolated by simple crystallization from hexane ethyl acetate and were fully characterized by NMR and HRMS analysis (included in the Supporting Information).

Scheme 1. Synthesis of the Potential Inhibitors.

With the target compounds in hand, we then proceeded to investigate their photo-cross-linking ability for HCA II. The enzyme (at a fixed concentration) was incubated for 15 min with each compound (20 μM) separately and then photoirradiated (30 min). The irradiated mixture was then subjected to gel electrophoresis under denaturing conditions. The gel was first visualized under UV and then stained with Coommassie blue. In both cases, the results were documented and then analyzed by Image J software.¹⁵ The gel pictures and the relative efficiency of cross-linking are shown in Figure 5.

Figure 5.

(A) SDS PAGE of purified HCA II cross-linked with the final compounds. C stands for control (only protein) and M stands for MW marker. Each compound (20 μM) has been used in each respective lane. (B) Image J analysis of the gel pictures to compare the relative efficiencies of cross-linking. L1-L18 denotes the corresponding lane numbers in (A). (C) Top: Relative cross-linking efficiencies of the control compound 13 and the two analogous compounds 2 and 3. Bottom: Image J analysis of the gel picture at the top.

Analysis of the gel pictures along with an overall image analysis clearly shows that all the 4-substituted benzene sulfonamides (1–3) showed the highest and also similar (considering the error range of ±5%) cross-linking efficiencies when compared with the control compound 13 (Figure 5C). Interestingly, the 3-aminosulfonamide (4) behaves similarly to the 4-susbtituted derivatives. Except for the benzoyl sulfonamide (8), which showed low but perceptible cross-linking efficiency, all other compounds including the 2-aminosulfonamide (5) showed insignificant cross-linking.

To check the selectivity and any off-targets, compounds 3 and 4 were incubated with the lysate of Escherechia coli cells overexpressed with HCA II.¹⁶ Sulfonamide 3 was found to be selective, while sulfonamide 4 was found to cross-link with other proteins to a significant extent (comparison of lanes 2 and 3 in Figure 6).

Figure 6.

Cross-linking of compounds 3 and 4 with cell lysate. Lane 1: molecular weight marker. Lane 2: compound 4. Lane 3: compound 3. The gel picture was taken under UV and then stained with Coomassie blue. The amount of compound used in each lane is 20 μM.

To validate the results based on cross-linking, we determined the IC₅₀ values of some of the compounds with strong and weak photo-cross-linking activity and compared these with those of the reference compound 13. The IC₅₀ values (shown along with the curves in Figure 7) also follow the same order as that observed from the gel-based assay. The K_i values for compound 3 showing the highest cross-linking efficiency were compared with those of compounds 5 and 6 with insignificant cross-linking. The trend remains similar. Incidentally, the reference compound 13 having a similar cross-linking efficiency to that observed for 3 also has a nanomolar K_i value (table in Figure 7).

Figure 7.

Determination of IC₅₀ and K_i values of some key compounds.

In conclusion, the present method offers a rapid way of initial screening of potential inhibitors, and based on the efficiency of photo-cross-linking, compounds can be selected for proceeding further, and weakly cross-linked or promiscuous inhibitors showing lack of selectivity can be discarded. To check whether the fluorescent photoreactive template works for other enzymes, derivatives with an appropriate selectivity functionality were made to target the penicillin-binding protein (PBP), metallo-β-lactamases like NDMs, and the fatty acid dehydratase enzymes HadAB and HadBC. In all cases, we could detect successful cross-linking demonstrating that the template has little effect on the binding efficiency of the selectivity hand. While the details of successful cross-linking with PBP and NDMs have already been published,^9,17 the cross-linking results with other enzymes will be reported after more elaborative studies.

Experimental Section

General Procedure

All the reactions under an inert atmosphere were conducted with oven-dried glassware with anhydrous solvents dried using standard methods and purified by distillation prior to use. All common reagents were of commercial grade unless otherwise specified. Thin-layer chromatography (TLC) was performed on aluminum-backed plates coated with Silica gel 60. A locally available ultraviolet light chamber was used as the TLC spot indicator. All new compounds were characterized using ¹H nuclear magnetic resonance (NMR) and ¹³C NMR spectroscopies. The NMR spectra were recorded using Bruker 400 MHz and 600 MHz spectrometers. Proton and carbon spectra were referenced internally to solvent signals using values of δ = 2.50 for proton and δ = 39.52 for carbon (middle peak) in DMSO-d₆, of δ = 7.26 for proton and δ = 77.16 for carbon (middle peak) in chloroform-d, and of δ = 2.05 for proton and δ = 206.26 and 29.84 for carbon (middle peak) in acetone-d₆. The following abbreviations have been used for NMR peak assignments: s = singlet, bs = broad singlet, d = doublet, t = triplet, p = pentet, m = multiplet, and dd = double of doublet. All biochemical experiments have been done as described in our previous papers.^18,17

Preparation of GABA-Carboxylic Acid (14)

The procedure for preparation of the GABA-carboxylic acid derivative is same as that reported earlier.⁹ To a solution of 4-azido-1,8-naphthalic anhydride (0.05 g, 0.2 mmol) in dry ethanol (7 mL), DMAP (0.002 g, 0.02 mmol) was added and stirred for 10 min. 4-Aminobutyric acid (GABA) (0.272 mmol) was added and the mixture was refluxed for 12 h. After cooling, the precipitated yellow solids were separated from the solution, washed with cold ethanol, and air-dried to furnish the azidonaphthalimide GABA-carboxylic acid. The compound was reprecipitated from the DCM–hexane mixture and washed with hexane to get the carboxylic acid as a yellow solid.

General Method for Preparation of the Bromoacetyl Derivative of Sulfonamides (15–19 and 21–23) and Carboxamide (20)

To a solution of sulfanilamide/carboxamide (0.35 g, 2.03 mmol) in dry THF (10 mL), K₂CO₃ (0.561 g, 4.06 mmol) was added and stirred for 30 min. It was cooled to 0 °C, and bromoacetyl chloride (0.2 mL, 2.44 mmol) was added dropwise to the reaction mixture and was stirred for 30 min at 0 °C. Water was added, and the mixture was extracted with EtOAc (50 mL × 2), washed with brine, dried over Na₂SO₄, and concentrated in vacuo to get the product as a white crystalline solid. The spectral data and other details are mentioned below.

Compound 15

White semisolid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.72 (s, 1H), 7.79 (d, J = 8.8 Hz, 2H), 7.74 (d, J = 8.8 Hz, 2H), 7.29 (s, 2H), 4.08 (s, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 171.5, 141.5, 138.5, 126.5, 119.2, 61.9. HRMS: Calcd for C₈H₉N₂O₃SBrNa (M + Na)⁺ 314.9415, found 314.9416.

Compound 16

White semisolid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.88 (t, J = 5.7 Hz, 1H), 7.77 (d, J = 8.3 Hz, 2H), 7.43 (d, J = 8.3 Hz, 2H), 7.32 (s, 2H), 4.36 (d, J = 5.9 Hz, 2H), 3.93 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.1, 143.8, 142.5, 127.6, 125.6, 61.5, 41.3. HRMS: Calcd for C₈H₉N₂O₃SBrNa (M + Na)⁺ 314.9415, found 314.9416.

Compound 17

White crystalline solid; mp 146–152 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.38 (t, J = 5.3 Hz, 1H), 7.74 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.30 (s, 2H), 3.82 (s, 2H), 3.41–3.23 (m, 2H), 2.80 (t, J = 7.1 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.1, 143.5, 142.1, 129.2, 125.8, 40.3, 34.5, 29.5. HRMS: Calcd for C₁₀H₁₃N₂O₃SBrNa (M + Na)⁺ 342.9728, found 342.9727.

Compound 18

White semisolid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.72 (s, 1H), 7.79 (d, J = 8.8 Hz, 2H), 7.74 (d, J = 8.8 Hz, 2H), 7.29 (s, 2H), 4.08 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.4, 144.8, 139.0, 129.8, 122.2, 121.0, 116.4, 30.3. HRMS: Calcd for C₈H₉N₂O₃SBrNa (M + Na)⁺ 314.9415, found 314.9411.

Compound 19

White crystalline solid; mp 149–152 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.76 (s, 1H), 8.05 (d, J = 8.2 Hz, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.70–7.55 (m, 3H), 7.35 (t, J = 7.7 Hz, 1H), 4.25 (s, 2H). ¹³C NMR (150 MHz, acetone-d₆) δ 165.4, 135.7, 133.9, 132.4, 128.8, 125.1, 123.4, 30.4. HRMS: Calcd for C₈H₉N₂O₃SBrNa (M + Na)⁺ 314.9415, found 314.9424.

Compound 20

White crystalline solid; mp >196 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.59 (s, 1H), 7.85 (d, J = 8.7 Hz, 2H), 7.64 (d, J = 8.6 Hz, 2H), 7.27 (s, 1H), 4.06 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.3, 167.4, 141.2, 128.9, 128.3, 118.7, 62.0. HRMS: Calcd for C₉H₉N₂O₂BrNa (M + Na)⁺ 278.9745, found 278.9743.

Compound 21

White semisolid. ¹H NMR (500 MHz, DMSO-d₆) δ 11.99 (s, 1H), 10.15 (s, 1H), 7.92 (d, J = 8.9 Hz, 2H), 7.83 (d, J = 8.9 Hz, 2H), 4.03 (s, 2H), 1.90 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ 171.8, 168.7, 143.1, 133.2, 128.7, 119.1, 61.9, 23.2. HRMS: Calcd for C₁₀H₁₁N₂O₄SBrNa (M + Na)⁺ 356.9521, found 356.9525.

Compound 22

White semisolid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.48 (s, 1H), 10.78 (s, 1H), 7.97–7.89 (m, 2H), 7.85 (d, J = 7.3 Hz, 2H), 7.77 (d, J = 8.8 Hz, 2H), 7.57 (t, J = 7.4 Hz, 1H), 7.45 (t, J = 7.7 Hz, 2H), 4.07 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.8, 165.3, 143.2, 133.2, 131.5, 129.2, 128.9, 128.6, 128.4, 119.1, 61.9. HRMS: Calcd for C₁₅H₁₃BrN₂O₄SK (M + K)⁺ 434.9416, found 434.9416.

Compound 23

White semisolid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.66 (s, 1H), 7.69 (d, J = 6.8 Hz, 4H), 6.83–6.53 (m, 4H), 4.06 (s, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 171.4, 157.9, 141.0, 138.8, 126.5, 119.0, 61.9. HRMS: Calcd for C₉H₁₁N₄O₃SBrNa (M + Na)⁺ 356.9633, found 356.9633.

Compound 24

Synthesis, NMR, and HRMS data reported earlier.¹⁴

General Method for Preparation of Sulfonamides 1–9

To a solution of azidonaphthalimide GABA-carboxylic acids (0.15 mmol) in dry DMF (5 mL) under N₂, anhydrous K₂CO₃ (0.18 mmol) was added and stirred for 30 min at room temperature. A solution of bromoacetyl derivatives of sulfonamide/carboxamide (0.18 mmol) in dry DMF (2 mL) was added, and stirring was continued for 10 h at room temperature. The reaction was quenched by adding water (30 mL), and the aqueous layer was extracted with EtOAc (30 mL × 2). The combined organic layers were washed with brine, aqueous NaHCO₃, and water, dried over anhydrous Na₂SO₄, and concentrated in vacuo. The yellowish brown gummy product was first precipitated from the acetone–hexane mixture to get the yellow precipitate, which was washed with hexane 2–3 times to furnish the target materials as yellow solids. The spectral and other details are mentioned below

Compound 1

Yellow solid; mp 150–152 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 10.35 (s, 1H), 8.52 (d, J = 6.4 Hz, 1H), 8.46 (d, J = 7.9 Hz, 1H), 8.41 (d, J = 7.6 Hz, 1H), 7.87–7.84 (m, 1H), 7.77 (m, 4H), 7.69 (d, J = 8.8 Hz, 1H), 7.25 (s, 2H), 4.65 (s, 2H), 4.11 (t, J = 6.9 Hz, 2H), 2.54 (t, J = 7.4 Hz, 2H), 2.00–1.95 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 172.1, 165.9, 163.4, 163.0, 142.8, 141.2, 138.7, 131.6, 131.5, 128.4, 128.3, 127.2, 126.8, 126.7, 123.5, 122.2, 118.8, 115.9, 62.4, 30.9, 22.9. IR (KBr, cm^–1): 3337, 2125, 1676, 1593, 1353, 1158, 1098, 840, 783. HRMS: Calcd for C₂₄H₂₀N₆O₇SNa (M + Na)⁺ 559.1012, found 559.1011.

Compound 2

Yellow solid; mp (d) 169–173 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.58 (t, J = 6.0 Hz, 1H), 8.44 (d, J = 7.2 Hz, 1H), 8.34 (m, 2H), 7.78 (m, 3H), 7.65 (d, J = 8.0 Hz, 1H), 7.41 (d, J = 8.2 Hz, 2H), 7.31 (s, 2H), 4.50 (s, 2H), 4.36 (d, J = 5.9 Hz, 2H), 4.07 (t, J = 6.9 Hz, 2H), 2.53 (m, 2H), 1.94 (p, J = 7.0 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.1, 167.1, 163.3, 162.9, 143.2, 142.7, 142.6, 131.5, 131.4, 128.2, 127.4, 127.7, 125.6, 123.4, 122.1, 118.1, 115.8, 62.3, 41.5, 30.9, 22.8. IR (KBr, cm^–1): 2117, 1741, 1658, 1542, 1280, 1153, 782, 679, 527. HRMS: Calcd for C₂₅H₂₂N₆O₇SNa (M + Na)⁺ 573.1168, found 573.1168.

Compound 3

Yellow solid; mp (d) 155–162 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.53 (d, J = 7.2 Hz, 1H), 8.48 (d, J = 8.0 Hz, 1H), 8.43 (d, J = 8.4 Hz, 1H), 8.07 (t, J = 5.6 Hz, 1H), 7.87 (t, J = 7.9 Hz, 1H), 7.74 (m, 3H), 7.37 (d, J = 8.2 Hz, 2H), 7.29 (s, 2H), 4.38 (s, 2H), 4.09 (t, J = 6.9 Hz, 2H), 2.78 (t, J = 7.2 Hz, 2H), 1.94 (p, J = 7.2 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.0, 166.7, 163.4, 162.9, 143.5, 142.8, 142.1, 131.6, 131.5, 129.1, 128.3, 127.2, 125.7, 123.5, 122.1, 118.1, 115.9, 112.4, 62.2, 34.7, 30.9, 22.8. IR (KBr, cm^–1): 3316, 2117, 1658, 1579, 1352, 1282, 1156, 714, 577. HRMS: Calcd for C₂₆H₂₄N₆O₇SNa (M + Na)⁺ 587.1325, found 587.1326.

Compound 4

Yellow solid; mp (d) 159–165 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.33 (s, 1H), 8.53 (d, J = 7.3 Hz, 2H), 8.48 (d, J = 8.0 Hz, 1H), 8.43 (d, J = 8.4 Hz, 1H), 8.13 (s, 1H), 7.86 (t, J = 7.9 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 6.6 Hz, 1H), 7.50 (d, J = 6.5 Hz, 2H), 7.37 (s, 5H), 4.64 (s, 2H), 4.11 (t, J = 6.9 Hz, 3H), 2.55 (t, J = 7.4 Hz, 3H), 2.01–1.84 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.2, 165.9, 163.4, 163.0, 144.7, 142.9, 138.7, 131.5, 129.5, 128.4, 127.3, 123.6, 122.2, 120.6, 118.3, 116.4, 116.0, 62.4, 31.0, 22.9. IR (KBr, cm^–1): 3264, 2124, 1693, 1650, 1587, 1351, 1155, 782, 589. HRMS: Calcd for C₂₄H₂₀N₆O₇S (M + H)⁺ 537.192, found 537.1252.

Compound 5

Yellow solid; mp (d) 181–183 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.14 (s, 1H), 8.49 (d, J = 7.2 Hz, 1H), 8.42 (d, J = 8.0 Hz, 1H), 8.38 (d, J = 8.4 Hz, 1H), 7.82 (m, 2H), 7.68 (t, J = 7.5 Hz, 2H), 7.47 (t, J = 7.6 Hz, 1H), 7.37 (d, J = 8.2 Hz, 1H), 4.89 (s, 2H), 4.10 (t, J = 6.5 Hz, 2H), 2.58 (t, J = 7.2 Hz, 2H), 2.03–1.91 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 172.1, 163.5, 163.1, 155.1, 142.9, 134.7, 133.3, 131.65, 131.6, 128.5, 128.4, 127.4, 126.7, 123.6, 122.3, 121.6, 118.3, 117.7, 116.0, 62.2, 30.9, 22.8. IR values (KBr, cm^–1): 2942, 2122, 1745, 1695, 1652, 1612, 1586, 1542, 1478, 1439, 1391, 1353, 1276, 1158, 1130, 996, 765, 588, 502. HRMS: Calcd for C₂₄H₂₀N₆O₇SNa (M + Na)⁺ 559.1012, found 559.1011.

Compound 6

Yellow solid; mp (d) 160–164 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.24 (s, 1H), 8.54 (d, J = 7.3 Hz, 1H), 8.49 (d, J = 8.0 Hz, 1H), 8.43 (d, J = 8.4 Hz, 1H), 7.90–7.80 (m, 4H), 7.76 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 8.6 Hz, 2H), 4.64 (s, 2H), 4.12 (t, J = 6.7 Hz, 2H), 2.55 (t, J = 7.4 Hz, 2H), 1.97 (p, J = 7.1 Hz, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 172.2, 167.3, 165.8, 163.4, 163.0, 142.8, 141.0, 131.6, 131.5, 129.1, 128.4, 128.4, 127.3, 123.5, 122.3, 118.4, 118.3, 116.0, 62.5, 31.0, 22.9. IR (KBr, cm^–1): 3411, 2123, 1691, 1652, 1592, 1253, 1196, 780, 630. HRMS: Calcd for C₂₅H₂₀N₆O₆Na (M + Na) 523.1342, found 523.1343.

Compound 7

Yellow solid; mp (d) 185–187 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 10.45 (s, 2H), 8.48 (d, J = 7.2 Hz, 1H), 8.41 (d, J = 8.0 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 7.86–7.80 (m, 3H), 7.74–7.65 (m, 3H), 4.65 (s, 2H), 4.09 (t, J = 6.8 Hz, 2H), 2.54 (t, J = 7.5 Hz, 2H), 1.97 (p, J = 7.2 Hz, 2H), 1.88 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ 172.1, 169.2, 166.1, 163.3, 162.9, 142.7, 142.5, 134.1, 131.5, 131.4, 128.8, 128.3, 128.2, 127.2, 123.4, 122.2, 118.7, 118.2, 115.8, 62.5, 30.9, 23.5, 22.9. HRMS: Calcd for C₂₆H₂₂N₆O₈SNa (M + Na)⁺ 601.1118, found 601.1115.

Compound 8

Yellow solid; mp (d) 189–192 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 12.46 (s, 1H), 10.45 (s, 1H), 8.54 (d, J = 7.3 Hz, 1H), 8.49 (d, J = 8.2 Hz, 1H), 8.43 (d, J = 8.3 Hz, 1H), 7.96–7.83 (m, 6H), 7.76 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 8.2 Hz, 2H), 7.58 (t, J = 6.9 Hz, 1H), 7.45 (t, J = 7.1 Hz, 2H), 4.64 (s, 2H), 4.11 (t, J = 6.7 Hz, 2H), 2.89 (s, 1H), 2.73 (s, 1H), 2.54 (t, J = 7.3 Hz, 2H), 2.01–1.93 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.1, 166.1, 163.5, 163.0, 142.8, 131.6, 131.5, 128.9, 128.3, 127.3, 123.6, 122.3, 118.6, 118.3, 116.0, 62.5, 22.9. IR (KBr, cm^–1): 3099, 2133, 1695, 1662, 1540, 1347, 1288, 1156, 833, 614, 559. HRMS: Calcd for C₃₁H₂₄N₆O₈SNa (M + Na)⁺ 663.1274, found 663.1273.

Compound 9

Yellow solid; mp (d) 181–183 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.31 (s, 1H), 8.52 (d, J = 7.3 Hz, 1H), 8.47 (d, J = 8.0 Hz, 1H), 8.42 (d, J = 8.4 Hz, 1H), 7.86 (t, J = 7.9 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.7 Hz, 2H), 7.63 (d, J = 8.8 Hz, 2H), 6.67 (s, 3H), 4.63 (s, 2H), 4.11 (t, J = 6.9 Hz, 2H), 2.54 (t, J = 7.4 Hz, 2H), 1.97 (p, J = 7.2 Hz, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 172.1, 165.8, 163.4, 163.0, 158.0, 142.8, 140.7, 139.2, 131.6, 131.5, 128.4, 128.3, 127.3, 126.6, 123.5, 122.2, 118.7, 118.3, 115.9, 62.4, 30.9, 22.9. IR (KBr, cm^–1): 3334, 2122, 1649, 1532, 1240, 1134, 823, 782, 633, 550. HRMS: Calcd for C₂₅H₂₂N₈O₇SNa (M + Na)⁺ 601.1230, found 601.1230.

Synthesis of 2-(5-(4-([2,2′:6′,2″-Terpyridin]-4′-yl)phenyl)-4-oxopentyl)-6-azido-1H-benzo[de]isoquinoline-1,3(2H)-dione (10)

The GABA-carboxylic acid derivative of 4-azido-1,8-naphthalimide (14) (0.11 g, 0.35 mmol) was dissolved in methanol, and NaHCO₃ (29 mg, 0.35 mmol) was dissolved in a minimum amount of water. To the solution of methanol, a NaHCO₃ solution was added. The mixture was stirred for 2 h, and the compound was lyophilized for 2 h to obtain the sodium salt of 14 as a yellow solid. To this solid in dry DMF, compound 24 (0.12 g, 0.35 mmol) was added. The reaction mixture was stirred for 12 h under a N₂ atmosphere at room temperature. The organic compound was extracted with ethyl acetate and washed with water to remove DMF. The yellow compound was further precipitated from the ethyl acetate–hexane layer. Yellow gummy solid; ¹H NMR (400 MHz, chloroform-d) δ 8.75–8.58 (m,7H), 8.55 (d, J = 8.0, 1H), 8.40 (d, J = 8.4, 1H), 7.87 (d, J = 6.0, 4H), 7.75–7.67 (m, 1H), 7.48–7.41 (m, 3H), 7.35 (t, J = 6.2, 2H), 5.16 (s, 2H), 4.26 (t, J = 7.0, 2H), 2.54 (t, J = 7.4, 2H), 2.15 (p, J = 7.1, 2H). ¹³C NMR (125 MHz, chloroform-d) δ 172.9, 164.2, 163.7, 156.3, 156.1, 149.9, 149.3, 143.6, 138.4, 137.0, 132.4, 131.9, 129.3, 129.0, 128.8, 127.6, 127.0, 124.5, 124.0, 122.7, 121.5, 119.0, 114.8, 66.0, 39.7, 32.1, 23.6. IR (KBr, cm^–1): 3404, 2931, 2123, 1708, 1352, 1272, 1111, 784, 726, 612. HRMS: Calcd for C₃₈H₂₇N₇O₄H (M + H)⁺ 646.2203, found 646.2201.

Synthesis of 4-(6-Azido-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-N-hydroxybutanamide (11)

To a solution of compound 14 (0.1 g, 0.31 mmol) in dichloromethane at 0 °C, ethyl chloroformate (0.04 g, 0.37 mmol) and N-methylmorpholine (0.041 g, 0.40 mmol) were added, and the mixture was stirred for 10 min. In another reaction vessel, an alcoholic solution of hydroxylamine was prepared by stirring hydroxylamine hydrochloride (0.043 g, 0.62 mmol) with potassium hydroxide (0.035 g, 0.62 mmol) in methanol for 15 min followed by filtration of the solution. The freshly prepared hydroxylamine in methanol was added to the previous reaction mixture in dichloromethane and stirred for 15 min. The solvent was removed under reduced pressure to obtain compound 11. Yellow gummy solid; ¹H NMR (400 MHz, methanol-d₄) δ 8.58 (d, J = 7.3, 1H), 8.55 (d, J = 8.0, 1H), 8.49 (d, J = 8.5, 1H), 7.80 (t, J = 7.8, 1H), 7.64 (d, J = 8.0, 1H), 4.18 (t, J = 7.0, 2H), 2.20 (t, J = 7.6, 2H), 2.02 (p, J = 7.7, 2H). ¹³C (125 MHz, methanol-d₄) δ 172.2, 165.5, 165.1, 145.3, 133.1, 133.1, 130.4, 130.0, 128.1, 125.6, 123.7, 119.8, 116.3, 40.7, 31.5, 25.4. IR (KBr, cm^–1): 3309, 2946, 2123, 1730, 1272, 1119, 733, 624. HRMS: Calcd for C₁₆H₁₃N₅O₄Na (M + Na)⁺ 362.0865, found 362.0868.

Synthesis of Compound 13

To a solution of 4-azido-1,8-naphthalic anhydride (0.1 g, 0.4 mmol) in dry ethanol (7 mL), DMAP (0.010 g, 0.08 mmol) was added and stirred for 10 min. Homosulfamine hydrochloride (0.098 g, 0.44 mmol) was added to the reaction mixture and refluxed for 12 h. After cooling, the precipitated yellow solids were separated from the solution, washed with cold ethanol, and were air-dried to furnish compound 13, and it was characterized without any further purification. Yellow solid; mp >195 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.51 (d, J = 7.3 Hz, 1H), 8.45 (d, J = 8.0 Hz, 1H), 8.40 (d, J = 8.4 Hz, 1H), 7.84 (t, J = 7.9 Hz, 1H), 7.78–7.68 (m, 1H), 7.53 (d, J = 8.2 Hz, 1H), 7.31 (s, 1H), 5.28 (s, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 163.3, 162.8, 143.2, 142.8, 141.3, 131.9, 131.8, 128.6, 128.4, 127.8, 127.3, 125.8, 123.5, 122.0, 117.9, 116.0, 42.7, 40.14. HRMS: Calcd for C₁₉H₁₃N₅O₄SH (M + H)⁺ 408.0766, found 408.0764.

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b01044.

• ¹H and ¹³C NMR spectra of all new compounds, HCA II isolation and electrophoresis procedure, and kinetics of cross-linking and K_i plots are included (PDF)

The authors declare no competing financial interest.